Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-14 to 2016-09-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and because there is ample experience and background data on this species and strain.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD)
- Females (if applicable) nulliparous and non-pregnant: yes (virgin)
- Age at study initiation: 6-7 wks
- Weight at study initiation: Males: 171-198 g; Females: 153-167 g
- Fasting period before study: Not specified
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in clear solid bottomed polysulfone cages measuring 59 x 38.5 x 20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5 x 26.6 x 18 cm with a stainless steel mesh lid and floor (Tecniplast – Gazzada S.a.r.l.). Each cage tray held absorbent material which was inspected and changed daily. After mating the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages measuring 42.5 x 26.6 x 18 cm (Tecniplast Gazzada S.a.r.l.) for the gestation period, birth and lactation. Suitable nesting material was provided and changed as necessary.
- Diet (e.g. ad libitum): commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: approximately 2 weeks

DETAILS OF FOOD AND WATER QUALITY: There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light - rooms lit by artificial light

IN-LIFE DATES: From: 2016-06-03 To: 2016-09-25

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is a possible route of exposure of the test item in man.
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The required amount of the test material was suspended in the vehicle. The formulation was prepared daily (concentrations of 10, 60, and 120 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is unstable in water, so corn oil was used as vehicle
- Concentration in vehicle: concentrations of 10, 60, and 120 mg/mL
- Amount of vehicle (if gavage): dose volume of 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method for formulation analysis was validated in RTC Study No. A2115 in the range from 5 to 150 mg/mL. Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure, in the range from 10 to 120 mg/mL, was acceptable (concentration and homogeneity). Final results for all levels were within the acceptability limits. Samples of the formulations prepared on Day 1 and last week (males and females) were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits. Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. A2115). The software used for this activity was Analyst 1.5.2.
Duration of treatment / exposure:
Males: dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy (Dose volumes were adjusted once per week for each animal according to the last recorded body weight).
Females: dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice (Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum).
Frequency of treatment:
Males and females: Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Low Dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Medium Dose
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
High Dose
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 50, 300 and 600 mg/kg/day were selected by the Sponsor, based on a previous, preliminary non GLP compliant study (RTC Study No.: Y0030).
- Rationale for animal assignment (if not random): rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Working days: twice a day, all parental animals were checked early in each working day in the morning and in the afternoon. Weekends and Public Holidays: a similar procedure was followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during treatment, each animal was observed and any clinical signs were recorded. Observations were performed approximately 0.5-1h after dosing each day.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to pairing and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after recording the terminal body weight
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females (females with viable litters) randomly selected from each group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after recording the terminal body weight
- Animals fasted: Yes
- How many animals: 5 males and 5 females (females with viable litters) randomly selected from each group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before commencement of treatment and once a week thereafter
- Dose groups that were examined: All animals
- Battery of functions tested: Parameters checked in Table [4] were examined

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 5).
- Male animals: All surviving parental males were sacrificed after completion of the mating period
- Maternal animals: All surviving parenteral females with live pups were sacrificed on Day 4 post partum. The females which did not give birth 25 days after positive identification of mating were killed shortly after.

HISTOPATHOLOGY: Yes (see table 5)
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test. The mean values, standard deviations and statistically analysis were calculated from the actual values in the computer without rounding off.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs were seen, on occasions, in animals treated at 600 mg/kg/day: salivation in males, rales and/or piloerection in females. A single male treated at 300 mg/kg/day showed rales on occasion. No compound related effects were seen in animals treated at 50 mg/kg/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were three unscheduled deaths at the high dose level (600 mg/kg/day): one male and one female were humanely killed on Day 4 and 6 of treatment (premating phase), respectively and one male was found dead on Day 22 of the mating phase.

Macroscopic changes such as red colour, pale firm areas or distended red/dark depressed areas in the stomach and reduced size of the thymus were confirmed at the histopathological evaluation that revealed marked mucosal ulceration in the non glandular region of the stomach and atrophy of thymus. The factors contributory to the death of these animals could be attributed to gastric lesions and thymus atrophy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the post coitum and post partum periods, slight but statistically significant reductions in body weight were seen in females receiving 300 and 600 mg/kg/day (up to approximately 10%). Reductions, significant at the statistical analysis, were seen in the body weight gain of males receiving 600 mg/kg/day on Day 8 before pairing, in males receiving 50 mg/kg/day at the end of the study, and in females receiving 300 and 600 mg/kg/day on Day 20 post coitum period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males receiving 600 mg/kg/day showed decreases in food consumption on Day 8 (-16%) statistically significant, and on Day 15 (-12%) of the premating period; a decrease (-20%) was also seen in females of the same group on Day 8 of premating period, compared to controls.Statistically significant decreases in food consumption were seen in females receiving 300 and 600 mg/kg/day during the gestation period (up to -16% and -19%, respectively) and on Day 4 post partum (-19%, not statistically significant, and -28%, respectively).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Changes as slight reticulocytosis and related changes in erythrocytes/haemoglobin or leucocytosis, observed in animals receiving 600 mg/kg/day, were considered of no toxicological relevance due to the absence of dose-relation and/or the minimal severity and/or incidence and/or presence of the same changes in control animals. No changes were seen in the coagulation parameter.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increases in alanine and aspartate aminotransferases, urea, creatinine, phosphorus and bile observed in single animals in all treated groups, not dose-related, were not considered to be suggestive of organ/tissue injury, due to the mild severity and the absence of histopathological related changes.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative thymus weights were decreased in males and females receiving 600 and 300 mg/kg/day statistically significant in the high dose group. This change was confirmed by the histopathological diagnosis of atrophy.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The most relevant changes observed at post mortem examination were dark/red colour or areas and/or thickened glandular and/or non glandular region of the stomach in high dose males and females and in low dose males, as well as an increased incidence of small thymus in most treated females, when compared with controls.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes were reported in the gastric tract (hyperplasia of the squamous non glandular epithelium, in some instances associated with ulceration and acute inflammation) and thymus (atrophy) of most treated animals of both sexes dosed at 300 and 600 mg/kg/day. No relevant changes were observed in the gastric tract and thymus of treated animals dosed at 50 mg/kg/day, when compared with controls.

The remaining lesions reported in control and/or treated animals such as atrophy of testes and epididymides were considered to be an expression of spontaneous and/or incidental pathology changes, commonly seen in this species and age under the laboratories experimental conditions.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 6. BODY WEIGHT (g) OF PREGNANT FEMALES – POST COITUM AND POST PARTUM PERIODS – GROUP MEAN DATA

Group(s)

 

Day of Phase

0!

7

14

20

1’’

4

1

(Control)

(n)

9

9

9

9

9

9

Mean

253.45

282.49

317.29

415.07

315.95

307.43

SD

10.89

7.05

9.51

11.34

12.21

10.09

 

2

(50 mg/Kg bw/day)

(n)

10

10

10

10

10

10

Mean

253.46

280.17

311.23

407.79

311.48

305.88

SD

10.14

8.17

9.65

23.98

9.48

17.18

 

3

(300 mg/Kg bw/day)

(n)

8

8

8

8

8

8

Mean

238.09*

261.83* ($)

294.37**

370.52**

297.11*

283.48

SD

17.62

18.42

17.58

22.25

20.89

29.79

 

4

(600 mg/Kg bw/day)

(n)

8

8

8

8

8

8

Mean

238.41*

260.24** ($)

295.42**

375.44**

283.22**

275.52* ($)

SD

9.71

6.27

6.38

17.52

14.47

24.57

! = Gestation phase; " = Post-partum phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

 

Table 7. BODY WEIGHT GAIN PER DAY° (g) OF PREGNANT FEMALES POST COITUM AND POST PARTUM PERIODS – GROUP MEAN DATA

Group(s)

 

Day of Phase

7!

14

20

4’’

1

(Control)

(n)

9

9

9

9

Mean

4.149

4.971

16.296

-2.840

SD

0.952

0.676

1.228

6.198

 

2

(50 mg/Kg bw/day)

(n)

10

10

10

10

Mean

3.815

4.438

16.092

-1.865

SD

0.789

0.929

3.260

5.930

 

3

(300 mg/Kg bw/day)

(n)

8

8

8

8

Mean

3.391

4.649

12.691*

-4.544

SD

1.416

1.488

1.807

4.758

 

4

(600 mg/Kg bw/day)

(n)

8

8

8

8

Mean

3.118

5.025

13.338*

-2.567

SD

1.407

0.925

2.461

6.559

! = Gestation phase; " = Post-partum phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

° = mean daily body weight gain over the previous period starting from Day 0 post coitum and Day 1 post-partum

 

Table 8. FOOD CONSUMPTION° (g/animal/day) OF PREGNANT FEMALES - POST COITUM AND POST PARTUM PERIODS – GROUP MEAN DATA

Group(s)

 

Day of Phase

7!

14

20

4’’

1

(Control)

(n)

9

9

9

9

Mean

18.36

21.82

26.82

29.57

SD

1.11

1.25

1.73

5.56

 

2

(50 mg/Kg bw/day)

(n)

10

10

10

10

Mean

18.29

20.30

25.37

30.61

SD

1.19

1.49

2.00

6.77

 

3

(300 mg/Kg bw/day)

(n)

8

8

8

8

Mean

15.68**

18.79**

22.58**

23.90

SD

2.19

2.03

2.53

4.26

 

4

(600 mg/Kg bw/day)

(n)

8

8

8

8

Mean

14.81**

18.77**

24.21*

21.36*

SD

1.19

0.99

2.46

8.05

! = Gestation phase; " = Post-partum phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

° = food consumed over the previous period starting from gestation Day 0 or post-partum Day 1

 

Table 9. HAEMATOLOGY - GROUP MEAN DATA

Parameter/units

 

Group(s)

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

Males

Reticulocytes (%)

Mean

2.124

2.208

2.380

3.095+

SD

0.273

0.268

0.456

0.393

n

5

5

5

4

 

Reticulocytes

(10^9/L)

Mean

167.56

174.44

188.92

231.98+

SD

22.23

18.80

30.80

22.26

n

5

5

5

4

Females

Mean Red Blood Cell Volume (fl)

Mean

61.30

60.34

59.76

65.56*

SD

1.72

2.65

1.53

2.99

n

5

5

5

5

 

Mean Corpuscular HB (pg)

Mean

20.12

19.92

19.80

21.82*

SD

0.87

0.79

0.97

1.26

n

5

5

5

5

 

White Blood Cell Count

(10^3/uL)

Mean

4.500

4.492

3.230

7.084+

SD

0.672

1.270

0.441

1.783

n

5

5

5

5

 

Lymphocytes (10^3/uL)

Mean

3.440

3.384

2.430

4.794*

SD

0.397

0.953

0.464

0.748

n

5

5

5

5

* = mean value of group is significantly different from control at p < 0.05

+ = mean value of group is significantly different from control at p < 0.01

 

Table 10. CLINICAL CHEMISTRY - GROUP MEAN DATA

Parameter/units

 

Group(s)

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

Males

Alkaline Phosphatase (U/L)

Mean

218.58

288.90

264.12

311.16*

SD

55.34

45.97

57.02

33.07

n

5

5

5

5

 

Alanine Amino Transferase

(U/L)

Mean

46.30

43.20

87.34

77.86*

SD

9.26

8.29

35.42

15.96

n

5

5

5

5

 

Bile Acids (µmol/L)

 

Mean

12.94

25.12

23.70

30.12*

SD

7.11

10.43

11.34

9.01

n

5

5

5

5

 

Globulin (g/dL)

Mean

2.44

2.46

2.26*

2.26*

SD

0.05

0.11

0.09

0.15

n

5

5

5

5

 

Inorganic Phosphorous (mg/dL)

Mean

6.192

5.946

7.424

9.088+

SD

1.171

0.488

0.984

1.980

n

5

5

5

5

Females

Alkaline Phosphatase (U/L)

Mean

219.36

200.46

251.46

377.68*

SD

57.95

86.72

18.56

97.73

n

5

5

5

5

 

Alanine Amino Transferase

(U/L)

Mean

47.96

62.78

58.12

83.90*

SD

4.15

44.17

10.01

18.75

n

5

5

5

5

 

Urea (mg/dL)

Mean

39.54

47.62

47.04

65.42+

SD

8.94

4.38

7.37

12.86

n

5

5

5

5

 

Creatinine ((mg/dL)

Mean

0.408

0.436

0.478*

0.536+

SD

0.38

0.025

0.031

0.038

n

5

5

5

5

 

Sodium (mmol/L)

Mean

140.74

139.32

139.84

139.02*

SD

0.85

3.68

0.97

0.87

n

5

5

5

5

* = mean value of group is significantly different from control at p < 0.05

+ = mean value of group is significantly different from control at p < 0.01

Table 11. ABSOLUTE ORGAN WEIGHTS (g) - GROUP MEAN DATA

Organ

Group:

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

Males

Adrenals

Number/Group

10

10

10

8

Mean

0.0528

0.0532

0.0566

0.0623

SD

0.0063

0.0040

0.0059

0.0082

Group diff atp<0.05

 

0.0068

0.0068

0.0072*

Group diff atp<0.01

 

0.0086

0.0086

0.0091*

Analysis of variance: F ratio = 4.36 Df = 3/ 34 F probability = 0.011

* indicates group mean is significantly different from control at level of significance shown

 

Heart

Number/Group

10

10

10

8

Mean

1.391

1.376

1.298

1.247

SD

0.099

0.060

0.114

0.126

Group diff atp<0.05

 

0.111

0.111

0.118*

Group diff atp<0.01

 

0.141

0.141

0.150

Analysis of variance: F ratio = 4.02 Df = 3/ 34 F probability = 0.015

* indicates group mean is significantly different from control at level of significance shown

 

Thymus

Number/Group

10

10

10

8

Mean

0.4056

3.645

0.3379

0.2645

SD

0.0763

0.0474

0.0478

0.0408

Group diff atp<0.05

 

0.0610

0.0610*

0.0647*

Group diff atp<0.01

 

0.0775

0.0775

0.0822*

Analysis of variance: F ratio = 10.03 Df = 3/ 34 F probability = 0.000

* indicates group mean is significantly different from control at level of significance shown

Females

Kidneys

Number/Group

10

10

10

9

Mean

1.770

1.706

1.593

1.654

SD

0.134

0.121

0.202

0.163

Group diff atp<0.05

 

0.173

0.173*

0.178

Group diff atp<0.01

 

0.220

0.220

0.226

Analysis of variance: F ratio = 2.27 Df = 3/ 35 F probability = 0.097

* indicates group mean is significantly different from control at level of significance shown

 

Thymus

Number/Group

10

10

10

9

Mean

0.2272

0.1933

0.1826

0.1442

SD

0.0568

0.0533

0.0564

0.0737

Group diff atp<0.05

 

0.0661

0.0661

0.0679*

Group diff atp<0.01

 

0.0838

0.0838

0.0861

Analysis of variance: F ratio = 3.06 Df = 3/ 35 F probability = 0.040

Note: a * indicates group mean is significantly different from control at level of significance shown

Data homogeneous by Bartlett's test (Dunnett's test)


Table 12. MICROSCOPIC OBSERVATIONS - GROUP INCIDENCE

Tissue with Diagnoses

 

Animals Affected

Animal Sex

Males

Females

Dosage Group

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

No. in group

10

10

10

8

10

10

10

9

Stomach

Number Examined

10

10

10

8

10

10

10

9

Mucosal Ulceration

 

0

0

1

2

0

0

0

1

Squamous Hyperplasia

 

0

0

4

-6

0

0

1

-9

Acute Inflammation

 

0

0

1

1

0

0

0

4

Mucosal Erosion

 

0

0

0

0

0

0

0

0

Necrosis

 

0

0

0

0

0

0

0

0

Oedema

 

0

0

0

0

0

0

0

0

Dilated Mucosal Gland

 

1

3

0

0

2

0

0

0

Chronic Inflammation

 

0

1

0

0

0

0

0

0

 

Thymus

Number Examined

10

10

10

8

10

10

10

8

Atrophy

 

0

0

1

-6

2

4

5

-7

Necrosis

 

0

0

0

0

0

0

0

0

Haemorrhage

 

0

0

0

2

1

0

0

0

Note: Entries flagged with a - (minus) are significantly higher than control at the 0.05 level using the Kolmogorov-Smirnov one-tailed test.

Table 13. TERMINAL BODY WEIGHT (g) - GROUP MEAN DATA

Group:

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

Males

Number/Group

10

10

10

8

Mean

408.46

394.74

389.91

385.64

SD

24.19

23.37

27.12

19.89

Group diff atp<0.05

 

26.38

26.38

27.98

Group diff atp<0.01

 

33.49

33.49

35.53

Analysis of variance: F ratio = 1.60             Df = 3/ 34             F probability = 0.205

* indicates group mean is significantly different from control at level of significance shown

Females

Number/Group

10

10

10

9

Mean

301.95

303.96

281.72

276.14

SD

11.20

20.10

31.14

25.89

Group diff atp<0.05

 

16.46

23.67

21.45*

Group diff atp<0.01

 

23.61

33.97

31.12

Analysis of variance: F ratio = 3.54             Df = 3/ 35             F probability = 0.024

* indicates group mean is significantly different from control at level of significance shown

Applicant's summary and conclusion

Conclusions:
Based on the results observed, the NOAEL (No Observed Adverse Effect Level) for systemic toxicity in the rat was determined to be 50 mg/Kg bw/day.
Executive summary:

In a key Guideline (OECD 422), combined repeated dose / reproductive developmental toxicity screening study, the test material in corn oil was administered via oral gavage to Sprague-Dawley rats (10/sex/dose) once daily at doses of 0, 50, 300, or 600 mg/Kg bw/day. Male rats were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day prior to necropsy. Female rats were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post-partum periods until Day 3 post-partum or the day prior to sacrifice.

 

Mortality was observed in the study as follows: Three unscheduled deaths in the high dose group (600 mg/Kg bw/day) - one male and one female humanely killed on Day 4/6 of the premating phase, respectively and one male found dead on Day 22 of the mating phase. Macroscopic changes such as red colour, pale firm areas or distended red/dark depressed areas in the stomach and reduced size of the thymus were confirmed by the histopathological evaluation that revealed marked mucosal ulceration in the non glandular region of the stomach and atrophy of thymus. Death of these animals could be attributed to gastric lesions and thymus atrophy.

 

Clinical signs such as salivation, rales and/or piloerection were seen, on occasion, in animals treated at 600 mg/Kg bw/day and in one animal dosed at 300 mg/Kg bw/day, while no treatment-related effects were seen in animals treated at 50 mg/Kg bw/day. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test material. No relevant differences were noted in motor activity and sensory reactivity to stimuli between control and treated groups.

 

Slight reductions in body weight (up to approximately 10%) were observed in females dosed at 300 and 600 mg/Kg bw/day during the post coitum and post-partum periods. Slight reductions in food consumption were seen in animals receiving 600 mg/Kg bw/day on Days 8 and/or 15 of the premating period, compared to controls. Decreases in food consumption were also seen in females receiving 300 and 600 mg/Kg bw/day during the gestation period and on Day 4 post-partum. No toxicological relevance was attributed to slight, not dose-related, changes observed in clinical pathology parameters.

 

A decrease (up to -9%) was observed in the terminal body weight of all treated males and in females receiving 300 and 600 mg/Kg bw/day, when compared to the controls. Reduction of thymus weights were seen in males and females receiving 300 and/or 600 mg/Kg bw/day. The most relevant changes observed at post mortem examination were dark/red colour or areas and/or thickened glandular and/or non glandular region of the stomach in 600 mg/Kg bw/day males and females and in 50 mg/Kg bw/day males, as well as an increased incidence of small thymus in most treated females, when compared with controls. Treatment-related changes were reported in the gastric tract (hyperplasia of the squamous non glandular epithelium, in some instances associated with ulceration and acute inflammation) and thymus (atrophy) of most treated animals of both sexes dosed at 300 and 600 mg/Kg bw/day. No relevant changes were observed in the gastric tract and thymus of treated animals dosed at 50 mg/kg/day, when compared with controls.

 

Based on the results obtained in this study, the dose level of 50 mg/Kg bw/day was determined to be the NOAEL (No Observed Adverse Effect Level) for systemic toxicity in the rat.